Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 19 disorders characterized by progressive wasting and weakness of pelvic and shoulder girdle muscles. Commonly, patients require wheelchair assistance, and individuals with some forms of LGMD may also have cardiac and respiratory muscle involvement. Modest improvements in a relatively limited set of muscles may dramatically improve patients' quality of life, but there is currently no effective treatment for LGMD. Gene therapy approaches may hold promise as future LGMD treatments. Over the last two decades, the feasibility of using gene therapy to treat muscular dystrophy in general, and LGMD in particular, has improved, due to important technical advancements made in vector design, production, and delivery. These advancements have almost exclusively been used to develop strategies aimed at replacing defective or missing genes underlying recessive disorders. However, gene replacement strategies are not feasible for treating dominant muscular dystrophies, including the 7 dominant forms of LGMD (called LGMD1). Instead, patients with dominant LGMD1 would likely benefit from reduction of their pathogenic alleles, which was historically not possible. Recently, RNA interference (RNAi) has emerged as a powerful tool to suppress dominant disease genes. This discovery has ushered in a new arm of gene therapy, in which therapeutic inhibitory microRNAs are engineered to reduce expression of toxic dominant disease genes, following delivery to affected tissues. The Specific Aims of this proposal were designed to test the feasibility of using RNAi to treat LGMD1A, which is caused by mutations in one allele of the myotilin (MYOT) gene. MYOT is an ideal target to demonstrate proof-of-principle for RNAi-mediated gene therapy of dominant LGMD1A because normal muscles are unaffected by its absence, and because an excellent LGMD1A mouse model is available for our studies. The former feature obviates the need to discriminate between mutant and normal alleles (which is feasible but more challenging than a non-allele- specific silencing approach) and therefore simplifies the strategy. Myotilin-directed microRNAs will be delivered to muscles of LGMD1A mice using adeno-associated viral vector (AAV) systems and delivery methods that have already been optimized for muscle gene transfer, including in clinical trials. The effects of microRNA-mediated MYOT knockdown on LGMD1A-associated histopathological and functional abnormalities will be assessed. These studies represent important first steps toward targeted RNAi gene therapy approaches for LGMD1A and may have implications beyond this proposal. Importantly, since dominant myopathies arise from mutations in at least 37 different genes that collectively affect ~1 in 2,400 to 3,200 individuals, our RNAi strategies could be adapted to broadly impact a large class of dominant muscle disorders. )) PUBLIC HEALTH RELEVANCE: Patients with dominant Limb Girdle Muscular Dystrophy Type 1A (LGMD1A) have one normal, and one mutated, copy of the myotilin gene. We propose that reducing mutant myotilin in muscles will improve muscular dystrophy in LGMD1A patients. In this proposal, we will investigate a strategy to reduce mutant myotilin using gene therapy.